Cyclopurines as Candidate Neurodegenerative DNA Lesions in Xeroderma Pigmentosum Studies in the past year have continued to make progress in understanding the biological significance of a novel class of oxidative DNA lesions called cyclopurines (cPu) that are formed in DNA as a result of the hydroxyl radical. These lesions are unique amongst oxidative DNA lesions in that they are specifically repaired by the nucleotide excision repair pathway. We have previously proposed that the accumulation of cyclopurine lesions on the transcribed strand of active genes is responsible for the neurodegeneration observed in patients with xeroderma pigmentosum who lack the capacity to carry out NER. In our previous work, we showed that a single cyclopurine can prevent binding of the TATA binding protein to a TATA box in vitro, and also strongly reduce gene expression in vivo. Katie Krone in our laboratory has now extended this work showing that a single cyclopurine lesion can block the binding of multiple sequence specific transcription factors, including CREB, heat-shock factor, and NF-kappa B. We are currently investigating the effect of the lesion on the structure-specific factor HMGI-Y. In addition to effects of cyclopurines on transcription factor binding, we have also investigated the possibility that RNA polymerase II can bypass cyclopurines in living mammalian cells. For this purpose, we created plasmid DNA constructs in which a single lesion was placed on the transcribed strand of a reporter gene, downstream from an intron. The lesion was placed in a unique restriction site to facilitate analysis of mutants. The lesion containing construct, or a lesion-free control, were transfected into NER -deficient cells, and isolated RNA analyzed by RT-PCR followed by restriction digestion and sequencing. Using this approach, we have obtained evidence that some abnormal (mutant) RNA transcripts can be produced when the polymerase bypasses the cyclopurine lesion in vivo. These results represent the first evidence that DNA lesions of the type that are repaired by the NER pathway can stimulate transcriptional mutagenesis. We are now writing a manuscript describing these results, and using the same methodology to study the bypass of other DNA lesions that are repaired by RNA pol II in vivo. Molecular Mechanisms of Neurodegeneration in Other DNA Repair Syndromes Mutations in the genes encoding several proteins involved in the detection of DNA damage lead to neurological disease. Work by other groups has shown that these proteins function as part of the DNA damage response in cells. Nearly all of this work has been focused on the DNA damage response in relation to cell-cycle checkpoints. Little is known about whether or even if postmitotic cells such as neurons can mount such a DNA damage response. In the past year, we have begun to address this question, and have found that neurons in culture can respond to the DNA damaging agent neocarzinostatin by phosphorylation the histone variant gamma H2AX, a well established DNA damage response in dividing cells. We are now actively working on extending this work to the nervous system of living animals. A Panel of Assays for Alcohol-Induced DNA Lesions Our working hypothesis in regard to the pathological effects of alcohol in the body is that these effects are mediated, at least in part, by damage to DNA. One specific DNA lesion that has been shown to be present in human alcoholics is N2-ethyl-2-deoxyguanosine (N2EtdG), which results from the reaction of acetaldehyde with deoxyguanosine. Dr. Jacob Theruvathu has developed a GC-MS assay for N2EtdG, as well as for a panel of other structurally different, oxidative DNA lesions. He has also succeeded in detecting this lesion in urine specimens from human volunteers. A Better Candidate for a mutagenic DNNA lesion in alcohol related cancer While N2EtdG appears useful as a marker for alcohol-related DNA damage, the biological effects of this lesion are not clear. We have therefore focused our attention on another acetaldehyde related DNA lesion, 1, N2-propanodeoxyguanosine (PdG). This lesion is repaired by NER, and has been shown to cause mutagenesis in mammalian cells. The formation of this lesion from acetaldehyde and deoxyguanosine has been reported to be stimulated by the basic amino acids lysine and arginine as are found in histones. We have found that PdG formation can also be stimulated by the polyamines spermine and spermidine. These highly basic molecules are present at high (mM) concentrations in cells. Polyamine synthesis is strongly correlated with cell division, and is elevated in cells undergoing active DNA synthesis. We therefore speculate that a high abundance of polyamines in rapidly dividing cells favors the formation of the mutagenic PdG adduct from AA, which may be relevant to the mechanisms of alcohol related gastrointestinal cancer. Ongoing studies are focused on the detection of this lesion in tissue samples in relation to alcohol abuse.